Herpes Simplex Virus Vaccines

ABSTRACT

The present invention is directed to Herpes simplex-2 viruses that may be used in vaccines to immunize patients against genital herpes.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to, and the benefit of, U.S.provisional application 61/288,836, filed on Dec. 21, 2009. This priorapplication is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is primarily concerned with vaccines that can beused to immunize patients against Herpes Simplex Virus type 2 (HSV-2)infections associated with chronic genital ulcers. The vaccine utilizesa replication defective HSV-2 virus that has been engineered to expresshigh levels of HSV-2 glycoprotein D antigen (gD2). In preferredembodiments, the HSV-2 virus also expresses one or more immunomodulatinggenes, such as IL15 and/or HSV-1 or HSV-2 major antigens such as gB orgC.

BACKGROUND OF THE INVENTION

Herpes Simplex Viruses (HSV) and HSV Infections

Herpes simplex virus 2 (HSV-2) is the primary cause of genital ulcerdisease. It can cause both an acute, productive infection and along-term latent infection characterized by unpredictable periodicrecurrences (66). Apart from causing lifelong, recurrent genital ulcers,HSV infections are a major concern in AIDS patients. It has beendocumented that genital HSV-2 infection triples the risk for sexuallyacquiring HIV infection (20), and in Africa, this increase in risk maycontribute to 25-35% of incident HIV infections (1).

Although the severity and duration of most symptomatic HSV primaryinfections can be reduced by oral or intravenous treatment withacyclovir, valacyclovir, or famciclovir, antiviral therapy neitherprevents the establishment of latent infection from primary infectionnor reduces subsequent recurrences (66). The continued spread of genitalherpes in the United States over the past two decades (19) and theincreasing incidence of HSV resistant to current antiviral medicationssuggest that there is a need for safe and efficacious vaccines againstHSV infections (31, 60). In addition, the finding that HSV suppressivetherapy leads to a significant reduction in levels of HIV in the genitalmucosa and plasma of women infected with both HSV-2 and HIV (52)suggests that an effective HSV vaccine may also have major implicationsin control of HIV infection (1, 31).

HSV-2 Glycoprotein D (gD2)

HSV glycoprotein D (gD) is one of the most predominant viral antigensexpressed on the surface of infected cells (21) and as well as on theviral envelope (24). gD is essential for the entry of the virus intocells and is a major target for neutralizing antibodies against HSVinfection (12, 49, 53). Moreover, gD is the predominant viral target forCD4⁺ T cells including CD4⁺ T cell cytotoxicity and CD8⁺ T cells inhuman and murine models of HSV infection (27, 28, 30, 34, 47, 65, 75).For these reasons, gD has been a major focus for HSV subunit vaccinedevelopment (32, 60).

In a phase 3 clinical trial, Stanberry, et al., showed that vaccinationwith recombinant gD from HSV-2 (gD2), in combination with adjuvant AS04,provided 73-74% efficacy in protecting against the development ofgenital herpes disease in HSV-seronegative women (62). No significantefficacy was observed, however, in men and in subjects who wereseropositive for HSV-1. Although gD2-specific humoral and CD4+ T cellresponses were detected in the immunized hosts, it is not clear whethergD2/AS04 was effective in eliciting a CD8+ T cell response (31, 32).This study suggests that there is a need for an HSV vaccine that elicitsa broader humoral, as well as CD4 and CD8 T-cell, response to both gD2and other HSV viral antigens (29, 31, 32).

Viral Vaccines

It is well documented that live viral vaccines capable of de novosynthesis of immunogens in the host induce a broader and more durableimmune response than vaccines consisting of only peptides or proteins.Various forms of replication-defective HSV and neuroattenuated,replication-competent mutants have been developed and tested aspotential in vaccines against HSV infection (U.S. Pat. No. 7,223,411;(18)).

Because both replication-defective viruses and neuroattenuated mutantscan co-replicate with wild-type virus or become replication-competent inthe context of wild-type virus, their use as a vaccine in humans poses asafety concern, particularly in individuals who harbor latent HSVinfection (33). The observation that replication-defective HSV-1 mutantscan reactivate the latent HSV-1 immediate-early promoter in the rodentbrain has raised additional safety concerns about the possibility ofsuch recombinants triggering outbreaks of productive viral infections inlatently infected individuals (63). Thus, a desirablereplication-defective recombinant HSV vaccine should not only possessthe ability to express a broad spectrum of virus-encoded antigens butshould also encode a unique function that can prevent lytic infection ofwild-type HSV when encountered within the same cells. Such a safetymechanism would minimize the potential outbreak of the vaccine viruscaused by the recombination of the vaccine vector with wild type virusin the host.

SUMMARY OF THE INVENTION

In general, the present invention is based upon the use of tetracyclinegene-switch technology (T-REx, Invitrogen) (73) and a dominant-negativemutant form of the HSV-1 UL9 polypeptide, e.g., UL9-C535C, to develop asafe and effective recombinant viral vaccine against HSV-2 infection.

In its first aspect, the invention is directed to areplication-defective, dominant-negative Herpes simplex virus 2 (HSV-2)recombinant virus. The genome of the virus has, at least, a firstsequence encoding a first HSV-2 glycoprotein D (gD2) operably linked toa first promoter and, preferably, a second sequence encoding a secondHSV-2 gD2 which is operably linked to a second promoter. The promoter(s)are operably linked to a first tetracycline operator (tet-O) sequenceand a second tet-O sequence respectively, each of which allowstranscription to proceed when free of tet repressor but which blockstranscription when bound by repressor. The genome also includes a thirdsequence which encodes, at least, a first dominant negative mutant formof the HSV-1 or HSV-2 UL9 protein linked to a third promoter and,preferably, a fourth sequence which encodes a second dominant negativeform of the HSV-1 or HSV-2 UL9 protein linked to a fourth promoter. Likethe first and second promoters, the third and fourth promoters are eachoperably linked to a tet-O sequence which, if bound by tet repressor,blocks transcription. In addition, the genome of the virus ischaracterized by the absence of a sequence encoding a functional ICP0protein. In order to enhance its antigenicity, the genome shouldpreferably also express immunomodulating genes, such as IL12 or IL15and/or HSV-1 or HSV-2 major antigens such as gB or gC.

The term “operably linked” refers to genetic elements that are joinedtogether in a manner that enables them to carry out their normalfunctions. For example, a gene is operably linked to a promoter when itstranscription is under the control of the promoter and thistranscription results in the production of the product normally encodedby the gene. A tet operator sequence is operably linked to a promoterwhen the operator blocks transcription from the promoter in the presenceof bound tet repressor and but allows transcription in the absence ofthe repressor. The term “recombinant” refers to a virus that has nucleicacid sequences that were, at some time, formed by the recombination ofnucleic acid sequences and sequence elements and the introduction ofthese recombined sequences into the virus or into an ancestor virus.

Preferably, the promoters used are those that have a TATA element andthe tet operator sequences linked to the promoters have two op2repressor binding sites joined together by between two and twentylinking nucleotides. The positioning of the operator sequence isimportant to achieve effective control over the promoter. Specifically,the first nucleotide in the operator sequence must be located betweensix and twenty-four nucleotides 3′ to the last nucleotide in the TATAelement. Structural sequences encoding, for example gD or adominant-negative mutant polypeptide of UL9, would lie 3′ to theoperator. Among specifically preferred promoters are the hCMV immediateearly promoter and HSV-1 or HSV-2 immediate early promoters. Especiallypreferred is the HSV-1 or HSV-2 ICP4 promoter.

In another aspect, the invention is directed to a vaccine that can beused prophylactically or therapeutically against HSV expression andwhich comprises one or more of the recombinant viruses described abovein unit dose form. The term “unit dose form” refers to a single drugadministration entity such as a tablet or capsule. Preferably the “unitdose form” will be a solution in which drug is dissolved at aconcentration that provides a therapeutic or prophylactic effect when aselected volume (unit dose) is administered to a patient by injectionand will be found within an injection vial. Based on the effective doseused in mice (2×10⁶ PFU), it is believed that the minimal effective dosein human should be about 1×10⁷ pfu. Thus, a unit dose should have atleast this amount of virus, with 1×10⁷-1×10⁹ pfu being typical. Vaccinesmay be stored in a lyophilized form and reconstituted in apharmaceutically acceptable carrier prior to administration.Alternatively, preparations may be stored in the vehicle itself. Thevolume of a single dose of the vaccine will vary but, in general, shouldbe between about 0.1 ml and 10 ml and, more typically, between about 0.2ml and 5 ml.

The invention also includes methods of immunizing patients against HSV-1or HSV-2 infection and the conditions resulting from such infection(e.g., genital Herpes ulcers) by administering to the patients thevaccines described above. The vaccines may also be given to patientsthat have been infected to prevent or reduce outbreaks of the virus. Anymethod for administering a vaccine to a patient which does not result inthe destruction of virus is compatible with the present invention.Generally, administration will be by parenteral means such as byintramuscular or intravenous injection. The dosage and scheduling ofadministration of vaccines can be determined using methods that areroutine in the art. The preparations may be administered in eithersingle or multiple injections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: FIG. 1A shows a schematic diagram of plasmids used forthe construction of dominant-negative and replication-defective HSV-2recombinants N2-C535C and CJ2-gD2. Plasmid pHSV-2/ICP0 is a plasmidcontaining the HSV-2 ICP0 sequences covering 268 bp upstream of theHSV-2 ICP0 open reading frame (grey box) to 40 bp downstream of the polyA signal of ICP0 coding sequences. pHSV2.ICP0-V was constructed byreplacing the Xho I-ICP0 DNA fragment containing sequences with an XhoI-containing multiple cloning sequence (MCS). pHSV2.ICP0-lacZ wascreated by inserting the LacZ gene (striped box) into the MCS region ofpHSV2.ICP0-V. p02lacZ-TOC535C was constructed by replacing the indicatedlacZ-containing fragment of pHSV2.ICP0-lacZ with DNA sequences encodingUL9-C535C (black box) under control of the tetO-containing hCMV majorimmediate-early promoter (line box, CMVTO). p02lacZTO-gD2.C535C wasconstructed by replacing the indicated SnaB I/Pst I fragment ofp02lacZTO-C535C with DNA sequences encoding gD2 gene (gradient box)under control of the tetO-containing HSV-1 immediate-early ICP4 promoter(open box, ICP4TO).

FIG. 1B shows a schematic diagram of genomes of wild-type HSV-2, anHSV-2 ICP0 null mutant (N2-lacZ), N2-C535C and CJ2-gD2. UL and USrepresent the unique long and unique short regions of the HSV-2 genome,respectively, which are flanked by their corresponding inverted repeatregions (open boxes). The replacement of both copies of the ICP0 codingsequences with the lacZ gene in N2-lacZ and with DNA sequences (1)encoding UL9-C535C under control of the tetO-bearing hCMV majorimmediate-early promoter in N2-C535C, and (2) encoding both UL9-C535Cand gD2 under the indicated tetO-bearing promoters in an oppositeorientation are shown below the expanded ICP0 coding sequences of theHSV-2 genome.

FIGS. 2A and 2B: These figures show high-level expression of gD2 andUL9-C535C following CJ2-gD2 infection of Vero cells. In FIG. 2A, Verocells in duplicate were either mock-infected or infected with wild-typeHSV-2, N2-lacZ, N2-C535C, or CJ2-gD2 at an MOI of 10 PFU/cell. Infectedcell extracts were prepared at 9 h post-infection. In FIG. 2B, Verocells were infected with wild type HSV-1 strain KOS, CJ9-gD, wild-typeHSV-2, or CJ2-gD2 at an MOI of 10 PFU/cell. Infected cell extracts wereprepared at 9 h post-infection. Proteins in infected cell extracts wereresolved on SDS-PAGE, followed by immunoblotting with polyclonalantibodies against HSV-1 gD (R45), UL9, or monoclonal antibodiesspecific for ICP27 and gB (Santa Cruz).

FIG. 3: FIG. 3 shows the regulation of gD2 and UL9-C535C expression bytetR in CJ2-gD2-infected VCEP4R-28 cells. VCEP4R-28 cells were seeded at5×10⁵ cells per 60-mm dish. At 40 h after seeding, cells in duplicatewere either mock-infected or infected with wild-type HSV-2 or CJ2-gD2 atan MOI of 10 PFU/cell in either the presence or the absence oftetracycline. Infected cell extracts were prepared at 9 h post-infectionfollowed by immunoblotting with polyclonal antibodies against HSV gD andUL9, and a monoclonal antibody specific for ICP27.

FIGS. 4A and 4B: These figures show the trans-dominant-negative effectof CJ2-gD2 on replication of wild-type HSV-2. In FIG. 4A, Vero cells intriplicate were infected with either wild-type HSV-2 strain 186 at anMOI of 2 PFU/cell, 186 at an MOI of 2 PFU/cell and CJ2-gD2 at an MOI of5 PFU/cell, or 186 at an MOI of 2 PFU/cell and N2-lacZ at an MOI of 5PFU/cell. In FIG. 4B, Vero cells were either singly infected withwild-type HSV-2 at an MOI of 5 PFU/cell, co-infected with 186 andCJ2-gD2 at an MOI of 5 PFU/cell for both viruses, or singly infectedwith 186 at an MOI of 15 PFU/cell, and co-infected with 186 at an MOI of15 PFU/cell and CJ2-gD2 at an MOI of 5 PFU/cell. Infected cells wereharvested at 18 h post-infection and viral titers were determined onVero cell monolayers. Viral titers are expressed as the mean +/−SD.Numbers on the top of the graph indicate the fold reduction in wild-typevirus yield between single infection and co-infection.

FIGS. 5A and 5B: These figures show the neurovirulence of wild-typeHSV-2, strain 186, N2-lacZ, N2-C535C, and CJ2-gD2 in BALB/c micefollowing intracerebral inoculation. Female BALB/c mice 4 to 6-weeks-oldwere randomly assigned to five groups of 8 mice each. Mice wereanesthetized with sodium pentobarbital and inoculated with either DMEM,25 PFU/mouse of wild-type HSV-2 strain 186, 1×10⁶ PFU/mouse of N2-lacZ,2.5×10⁶ PFU/mouse of CJ2-gD2 or N2-C535C through intracerebral injectioninto the left frontal lobe of the brain in a volume of 20 μl at a depthof 4 mm. Mice were examined for signs and symptoms of illness for 35days after inoculation. FIG. 5A shows disease score at various daysafter injection and FIG. 5B shows the percetage of mice surviving.

FIGS. 6A and 6B: FIGS. 6A and 6B are concerned with the induction ofgD2-specific antibodies and HSV-2-neutralizing responses. Female 4- to6-week-old BALB/c mice were either sham-immunized with DMEM (n=7, 6, 8,8) or immunized with CJ2-gD2 (n=7, 6, 8, 8), N2-C535C (n=7, 8, 6), orCJ9-gD (n=6, 8, 6) at a dose of 2×10⁶ PFU/mouse, and boosted 2 weekslater. Blood was obtained from the tail veins of mice 4-5 weeks afterprimary immunization. In FIG. 6A, serum from an individual group of micewas pooled and heat-inactivated. HSV-2-specific neutralizing antibodytiters were determined. The results represent average titers±SEM. InFIG. 6B, sera from sham-immunized, CJ2-gD2-, N2-C535C-, orCJ9-gD-immunized mice were incubated with cell extract prepared fromU2OS cells transfected with gD2-expressing plasmid p02.4TO-gD2. gD/mouseIgG-specific complexes were precipitated with Protein A, resolved onSDS-PAGE, and probed with a gD-specific polyclonal antibody, R45.

FIGS. 7A-7D: These figures are concerned with the induction ofHSV-2-specific CD4⁺ and CD8⁺ T-cell responses in CJ2-gD2-immunized mice.Female BALB/c mice were either sham-immunized or immunized with CJ2-gD2at 2×10⁶ PFU per mouse twice at 2-week interval. In FIGS. 7A and 7B,sham-immunized and immunized mice were either mock-infected or infectedwith wild-type HSV-2 s.c. at a dose of 1×10⁴ PFU/mouse at 9-10 weekspost boost immunization (n=3). The CD4⁺ and CD8⁺ T cell responses wereanalyzed on day 5 post-challenge by IFN-γ ELISPOT assays withindividually purified CD4⁺ and CD8⁺ T cells isolated from the mousespleen using Dynal mouse CD4- and CD8-negative kits. In FIGS. 7C and 7D,sham-immunized and CJ2-gD2 immunized mice were mock-infected or infectedwith wild-type HSV-2 at 5-6 weeks post boost immunization followed byIFN-γ ELISPOT assays on day 4 post-infection (n=3). The number of IFN-γspot-forming cells (SFC) was expressed as the mean±SEM per million CD4⁺or CD8⁺ T cells.

FIG. 8: FIG. 8 shows the reduction of challenge HSV-2 vaginalreplication in mice immunized with CJ2-gD2. Female 4- to 6-week-oldBALB/c mice were randomly assigned to 4 groups of 10 mice each. Micewere either mock-immunized with DMEM or immunized with CJ2-gD2,N2-C535C, or CJ9-gD at a dose of 2×10⁶ PFU/mouse. Mice were boostedafter 2 weeks. At 5 weeks, mice were pretreated with medroxyprogesteroneand challenged intravaginally with 5×10⁵ PFU of HSV-2 strain G. Vaginalswabs were taken on days 1, 2, 3, 5, and 7 post-challenge. Infectiousviruses in swab materials were assessed by standard plaque assay on Verocell monolayers. Viral titers are expressed as the mean±SEM inindividual vaginal swabs.

FIGS. 9A and 9B: These figures show the prevention of HSV-2 disease inmice immunized with CJ2-gD2. After challenge with wild-type HSV-2,individual mice described in the legend of FIG. 8 were observed during a21-day follow-up period for the incidence of genital and disseminatedHSV-2 disease (FIG. 9A) and survival (FIG. 9B) using the followingscale: 0=no sign, 1=slight genital erythema and edema, 2=moderategenital inflammation, 3=purulent genital lesions and/or systemicillness, 4=hind-limb paralysis, 5=death.

FIG. 10: FIG. 10 shows the HSV-1 UL9-C535C coding sequence (SEQ IDNO:2). UL9-C535C consists of UL9 amino acids 1-10, a Thr-Met-Glytripeptide, and amino acids 535 to 851 of UL9 (see Yao, et al. (69)).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the concept of using tetracyclinegene-switch technology and a dominant-negative mutant polypeptide ofHSV-1 UL9 to develop an HSV recombinant virus which is replicationdefective and capable of inhibiting wild-type HSV infections(dominant-negative). CJ9-gD is a prototype dominant-negative,replication defective HSV-1 recombinant virus and expresses high-levelsof HSV-1 major antigen glycoprotein D (gD) independent of HSV viral DNAreplication (7). In its most preferred form, the present invention usesa dominant-negative and replication-defective HSV-2 recombinant(CJ2-gD2) that encodes 2 copies of the HSV-2 gD (gD2) gene, driven bythe tetO-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2expresses gD2 as efficiently as wild-type HSV-2 and can exert a powerfultrans-inhibitory effect on the replication of wild type HSV-2 inco-infected cells. Immunization with CJ2-gD2 elicits effectiveHSV-2-specific neutralizing antibody as well as T-cell responses, andoffers a complete protection against intravaginal infection by wild-typeHSV-2 in mice.

CJ2-gD2 is a more effective vaccine than CJ9-gD in protection againstwild-type HSV-2 genital infection and disease. Furthermore,intracerebral injection of a high dose of CJ2-gD2 causes no mortality ormorbidity in mice. Collectively, these observations suggest that CJ2-gD2has advantages over traditional replication-defective virus vaccines andHSV-2 subunit vaccines in protecting against HSV-2 genital infection anddisease in humans.

The Tet Operator/Repressor Switch and Recombinant DNA

The present invention is directed to, inter alia, viruses having geneswhose expression is regulated by the tetracycline operator and repressorprotein. Methods that can be employed to make recombinant DNA moleculescontaining these elements and DNA sequences have been previouslydescribed (see U.S. Pat. No. 6,444,871; U.S. Pat. No. 6,251,640; andU.S. Pat. No. 5,972,650) and plasmids which contain thetetracycline-inducible transcription switch are commercially available(T-REx™, Invitrogen, CA).

An essential feature of the DNA of the present invention is the presenceof genes that are operably linked to a promoter, preferably having aTATA element. A tet operator sequence is located between 6 and 24nucleotides 3′ to the last nucleotide in the TATA element of thepromoter and 5′ to the gene. Virus may be grown in cells that expressthe tet repressor in order to block gene transcription and allow viralreplication. The strength with which the tet repressor binds to theoperator sequence is enhanced by using a form of operator which containstwo op2 repressor binding sites (each such site having the nucleotidesequence: TCCCTATCAGTGATAGAGA (SEQ ID NO:1)) linked by a sequence of2-20, preferably 1-3 or 10-13, nucleotides. When repressor is bound tothis operator, very little or no transcription of the associated genewill occur. If DNA with these characteristics is present in a cell thatalso expresses the tetracycline repressor, transcription of the genethat can prevent viral infection will be blocked by the repressorbinding to the operator and replication of the virus will occur.

Selection of Promoters and Genes

During productive infection, HSV gene expression falls into three majorclasses based on the temporal order of expression: immediate-early (α),early (β), and late (γ), with late genes being further divided into twogroups, γ1 and γ2. The expression of immediate-early genes does notrequire de novo viral protein synthesis and is activated by thevirion-associated protein VP16 together with cellular transcriptionfactors when the viral DNA enters the nucleus. The protein products ofthe immediate-early genes are designated infected cell polypeptidesICP0, ICP4, ICP22, ICP27, and ICP47 and it is the promoters of thesegenes that are preferably used in directing the expression of therecombinant genes discussed herein.

ICP0 plays a major role in enhancing the reactivation of HSV fromlatency and confers a significant growth advantage on the virus at lowmultiplicities of infection. ICP4 is the major transcriptionalregulatory protein of HSV-1, which activates the expression of viralearly and late genes. ICP27 is essential for productive viral infectionand is required for efficient viral DNA replication and the optimalexpression of viral γ genes and a subset of viral β genes. The functionof ICP47 during HSV infection appears to be to down-regulate theexpression of the major histocompatibility complex (MHC) class I on thesurface of infected cells.

The full length sequence of the HSV-1 genome sequence of the codingregion of the HSV-1 UL9-C535C is shown in FIG. 10 (SEQ ID NO:2). Theother sequences described for use in recombinant viruses are all wellknown in the art. For example the full length genomic sequence for HSV-1may be found as GenBank sequence X14112. The HSV-1 ICP4 sequence may befound as GenBank number X06461; HSV-1 glycoprotein D may be found asGenBank sequence J02217; HSV-2 glycoprotein D may be found as GenBanknumber K01408; and the HSV-1 UL 9 gene as GenBank sequence M19120 (allof which arc incorporated by reference herein in their entirety).

Inclusion of Tet Repressor and Making of Virus

Sequences for the HSV ICP0 and ICP4 promoters and for the genes whoseregulation they endogenously control are well known in the art (43, 44,56) and procedures for making viral vectors containing these elementshave been previously described (see US published application2005-0266564). These promoters are not only very active in promotinggene expression, they are also specifically induced by VP16, atransactivator released when HSV-1 or HSV-2 infects a cell.

Once appropriate DNA constructs have been produced, they may beincorporated into HSV-2 virus using methods that are well known in theart (see generally Yao et al. (68)).

Immunization Methods

The viruses described herein will be used to immunize individuals and/orpatients, typically by injection as a vaccine. The vaccine may be usedboth prophylactically to prevent HSV-1 or HSV-2 infection ortherapeutically to reduce the severity of an HSV-1 or HSV-2 infectionthat has already occurred. In order make a vaccine, the viruses can besuspended in any pharmaceutically acceptable solution including sterileisotonic saline, water, phosphate buffered saline, 1,2-propylene glycol,polyglycols mixed with water, Ringer's solution, etc. The exact numberof viruses to be administered is not crucial to the invention but shouldbe an “effective amount,” i.e., an amount sufficient to elicit animmunological response strong enough to inhibit HSV infection. Ingeneral, it is expected that the number of viruses (PFU) initiallyadministered will be between 1×10⁷ and 1×10¹⁰.

The effectiveness of a dosage, as well as the effectiveness of theoverall treatment can be assessed using standard immunological methodsto test for the presence of antibodies effective at attacking HSV.Immunological injections can be repeated as many times as desired.

EXAMPLES

The current example describes the creation of an HSV-2 recombinant virusand tests to determine its immunological effects.

1. Materials and Methods

Cells

African Green Monkey Kidney (Vero) cells and the osteosarcoma line U2OScells were grown and maintained in Dulbecco's Modified Eagle's Medium(DMEM; Sigma Aldrich) supplemented with 10% fetal bovine scrum (FBS) inthe presence of 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate(GIBCO, Carlsbad, Calif.) (71). U2OS cells are able to complementfunctionally for the HSV-1 ICP0 deletion (71). U2CEP4R11 cells aretetR-expressing U2OS cells that were maintained in DMEM plus 10% FBS andhygromycin B at 50 μg/m1 (73). VCEP4R-28 cells are tetR-expressing Verocells that were maintained in DMEM plus 10% FBS and hygromycin B at 50μg/ml (73).

Plasmids

Plasmid pHSV2/ICP0 is a pUC19 derived plasmid that encodes the PCRamplified HSV-2 ICP0 sequences covering 268 bp upstream of the HSV-2ICP0 open reading frame (ORF) to 40 bp downstream of the poly A signalof ICP0 coding sequences. pHSV2.ICP0-V is an HSV-2 ICP0 cloning plasmid,derived from plasmid pHSV-2/ICP0, by replacing the Xho I-ICP0 DNAfragment containing sequences 25 bp upstream of the initiation codon ofICP0 to 397 bp upstream of the stop codon of ICP0 ORF with a XhoI-containing multiple cloning sequence (MCS). Plasmid pHSV2.ICP0-lacZwas created by inserting HindIII-Not I-LacZ gene-containing fragment ofpcDNA3-lacZ into pHSV2.ICP0-V at the Hind III-Not I sites.pcmvtetO-UL9C535C is a plasmid encoding UL9-C535C under control of thetetO-containing hCMV immediate-early promoter (68). p02lacZ-TOC535C,expressing UL9-C535C driven by the tetO-containing hCMV majorimmediate-early promoter (FIG. 1A), was constructed by replacing theEcoR I/Age I-lacZ containing fragment of pHSV2.ICP0-lacZ with the EcoRI/Hind III-hcmvtetO-UL9C535C containing fragment of pcmvtetOUL9-C535C(69).

pAzgD-HSV-2 is an HSV-2 gD2-encoding plasmid kindly provided by Dr.Patricia Spear (Northwestern University). pICP4TO-hEGF expresses humanepidermal growth factor under control of the tetO-bearing HSV-1immediate-early ICP4 promoter, which consists of HSV-1 ICP4 promotersequence from −377 bp to −19 bp relative to the transcriptional startsite of ICP4 gene. Similar to the tetO-bearing hCMV majorimmediate-early promoter in plasmid pcmvtctO-hEGF (73), thetetO-containing ICP4 promoter contains two tandem copies of tetoperators at 10 bp downstream of the ICP4 TATA element, TATATGA. Thus,like pcmvtetO-hEGF, hEGF-expression from pICP4TO-hEGF can be tightlyregulated by tetracycline in the presence of tetR, and insertion of thetetO has no effect on the ICP4 promoter activity in the absence of tetR.An additional unique feature associated with the tetO-bearing ICP4promoter in pICP4TO-hEGF is the absence of the ICP4 DNA binding sequenceATCGTCCACACGGAG (SEQ ID NO:3), which spans the transcription initiationsite of ICP4 gene (51) in the wild-type ICP4 promoter. Thus, unlike thewild-type ICP4 promoter that is subject to auto-regulation by ICP4 (16,57), the tetO-bearing ICP4 promoter in pICP4TO-hEGF will not besuppressed by the HSV-1 major-regulatory protein ICP4.

To clone gD2 under the control of the tetO-containing ICP4 promoter, wefirst constructed plasmid p02ICP4-TO by cloning the Sma I-Bam HItetO-containing ICP4 promoter in pICP4TO-hEGF into pHSV2.ICP0-V into theMCS of the vector. p02.4TO-gD2 is a p02ICP4-TO derived plasmid thatencodes gD2 gene of pAzgD-HSV-2 under control of the tetO-bearing ICP4promoter.

p02lacZTO-gD2.C535C, a plasmid encoding UL9-C535C under the control ofthe tetO-bearing hCMV immediate-early promoter with a 5′ truncation at−236 bp of the hCMV promoter and the gD2 gene under control of thetetO-ICP4 promoter (FIG. 1A), was created by replacing the SnaB I/Pst Ifragment of p021acZTO-C535C with a Hind III/Pst I-gD2-containingfragment of p02.4TO-gD2. In p02lacZTO-gD2.C535C, the transcription ofUL9-C535C gene and gD2 gene are in an opposite orientation.

Viruses

Wild-type HSV-2, strains 186 and G, were propagated and plaque-assayedon Vero cells. N2-lacZ is a HSV-2 ICP0 null mutant encoding the Lac Zgene under the control of HSV-2 ICP0 promoter, in which both copies ofthe ICP0 gene are replaced by the Lac Z gene in pHSV2.ICP0-lacZ throughhomologous recombination by transfecting U2OS cells with NheI-linearized pHSV2.ICP0-lacZ followed by HSV-2 superinfection aspreviously described (74). The replacement of the ICP0 gene with the LacZ gene at the ICP0 locus was confirmed by PCR analysis of N2-lacZ viralDNA with the primers that flank the ICP0 gene and primers specific forthe lac Z gene (41, 74).

N2-C535C is a derivative of N2-lacZ, in which both copies of the Lac Zgene are replaced with DNA sequences encoding UL9-C535C under control ofthe tetO-containing hCMV promoter in plasmid p02lacZ-TOC535C (FIG. 1B).In brief, U2CEP4R11 cells were co-transfected with the linearizedp02lacZ-TOC535C and infectious N2-lacZ viral DNA by Lipofectamine 2000.Progeny of the transfection were screened for the recombinationalreplacement of the lacZ genes of N2-lacZ with the DNA sequencecontaining the cmvtetOUL9-C535C by standard plaque assays. Plaques werestained with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) 96hr postinfection. White plaques, reflecting the replacement of bothcopies of the lacZ gene by the UL 9-C535C DNA-encoding sequence, wereisolated. One of the isolates, designated N2-C535C, yielded uniformlywhite plaques after four rounds of plaque purification.

CJ2-gD2 is constructed by replacing both copies of the Lac Z gene at theICP0 locus in N2-lacZ with DNA sequences encoding UL9-C535C under thetetO-bearing hCMV major immediate-early promoter and gD2 under thecontrol of the tetO-containing HSV-1 ICP4 promoter (FIG. 1B), whichconsists of HSV-1 ICP4 promoter sequence from −377 bp to −19 bp relativeto the transcriptional start site of ICP4 gene (71).

SDS-PAGE and Western Blot Analysis

Vero cells seeded in 60 mm dishes at 7.5×10⁵ cells/dish weremock-infected or infected with indicated viruses at an MOI of 10PFU/cell. Cell extracts were prepared at 9 h or 16 h post-infection(72). Proteins in the cell extract were resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (9% acrylamide),transferred to polyvinylidene difluoride (PVDF) membranes, and probedwith either polyclonal antibodies against HSV-1 gD (R45, a gift of Drs.Gary H. Cohen and Roselyn J Eisenberg), UL9 (a gift of Mark Challberg),or monoclonal antibodies specific for ICP27 and gB (Santa CruzBiotechnology, Santa Cruz, Calif.).

Mice

Female BALB/c mice 4-6 weeks of age were purchased from Charles RiverLaboratories (Wilmington, Mass.). Mice were housed in metal cages atfour mice per cage and maintained on a 12 h-light/dark cycle. Mice wereallowed to acclimatize to the housing conditions for 1 week prior toexperimentation. All animal experiments were conducted according to theprotocols approved by Harvard Medical Area Standing Committee on Animalsand the American Veterinary Medical Association.

Immunization and Challenges

BALB/c mice were randomly divided into several groups and the hair ontheir left rear flank was trimmed. Mice were either vaccinated with2×10⁶ PFU/mouse of CJ2-gD2, N2-C535C, CJ9-gD, or mock-vaccinated withDMEM in a volume of 30 μl s.c. in the left rear flank using a 1-mlsyringe fitted with a 27-gauge needle. Mice were boosted after 2 weeksand challenged with wild-type HSV-2 strain G 3 weeks after secondaryimmunization. Five days prior to challenge, mice were injected s.c. inthe neck ruff with medroxyprogesterone (SICOR Pharmaceuticals, Inc.,Irvine, Calif.) at 3 mg per mouse in a volume of 20 μl (7, 50). Forintravaginal challenge, mice in all groups were anesthetized, preswabbedwith a calcium alginate swab (Sterile urethro-genital calcium alginatetipped applicator, Puritan Medical Products company LLC, Guilford, Me.USA) and inoculated intravaginally with 20 μl of culture mediumcontaining 5×10⁵ PFU (50 LD₅₀) of HSV-2 strain G (50). Animals were kepton their backs with their rear part elevated under the influence ofanesthesia for 30-45 min post-infection.

Acute Infection Assays and Clinical Observations

On days 1, 2, 3, 5, and 7 post-challenge, vaginal mucosac were swabbedwith calcium alginate (7). Infectious viruses in swab materials wereassessed by standard plaque assay on Vero cell monolayers. Followingchallenge with wild-type HSV-2, mice were assessed daily during a 21-dayfollow-up period for signs of genital lesions and systemic illness. Theseverity of disease were scored as follows: 0=no sign of herpeticinfection, 1=slight genital erythema and edema, 2=moderate genitalinflammation, 3=purulent genital lesions and/or systemic illness,4=hind-limb paralysis, and 5=death (8, 50).

Detection of HSV-2-Specific Neutralizing Antibodies

Blood was collected from tail veins of immunized and mock-immunized mice4 weeks after primary immunization. Neutralizing serum antibody titerswere determined as previously described in the presence of complement(5-7) with 250 PFU of wild-type HSV-2 strain 186. The neutralizingantibody titer was expressed as the final serum dilution required toachieve a 50% reduction in HSV PFU relative to the HSV PFU obtained inmedium plus complement alone.

Immunoprecipitation

U2OS cells seeded at 7.5×10⁶ cells per 100-mm dish were mock-transfectedor transfected with 10 μg of p02.4TO-gD by lipofectamine 2000 at 24 hpost-seeding. Cell extracts were prepared at 48 h post-transfection(72). Immunoprecipitations were performed by mixing 10 μl of pooledserum collected from mock-immunized and immunized mice with 70 μl ofcell extracts prepared above. The gD/mouse IgG-specific complexes wereprecipitated with Protein A (Pierce Classic IP kit, PierceBiotechnology, Rockford, Ill.), resolved on SDS-PAGE and probed with therabbit anti-gD-specific polyclonal antibody, R45, following by reactingwith HRP-conjugated goat-anti-rabbit IgG (Santa Cruz Biotechnology,Santa Cruz, Calif.).

IFN-γ ELISPOT Assays

Female BALB/c mice were sham-immunized with DMEM or immunized withCJ2-gD2 at a dose of 2×10⁶ PFU/mouse twice at 2-weeks apart. At 5 to 10weeks post second immunization, sham-immunized and CJ2-gD2-immunizedmice were mock-challenged or challenged with wild-type HSV-2 strain 186s.c. at a dose of 1×10⁴ PFU/mouse. Splenocytes were isolated fromindividual groups of mice (n=3) on days 4 or 5 post-challenge. The CD4⁺and CD8⁺ T cell ELISPOT assay was carried out as previously described(42). In brief, CD4⁺ and CD8⁺ T cells were isolated from splenocytesusing Dynal mouse CD4- or CD8-negative isolation kits and seeded inquadruplicate in a 96-well filtration plate pre-coated with anti-mouseIFN-γ specific monoclonal antibody (AN18) at 7.5×10⁴ or 1.5×10⁵cells/well. After incubation at 37° C. for 20 h, wells were washed,reacted with biotinylated IFN-γ specific monoclonal antibody (R4-6A2,Mabtech) at room temperature, and incubated with Streptavidin-AlkalinePhosphatase (Mabtech). The IFN-γ spot-forming cells were detected byaddition of BCIP/NBT substrate. Spots were counted in a dissectingmicroscope and the number of IFN-γ spot-forming cells (SFC) wasexpressed as the mean±SEM per million CD4⁺ or CD8⁺ T cells.

Quantitative Real-Time PCR

The lower lumbar and sacral part of the spinal column including spinalcord and dorsal root ganglia were collected 16 days after boostimmunization or 21 days after intravaginal challenge with 5×10⁵ PFU ofHSV-2 strain G from 9 or 10 mice that had been either immunized withCJ2-gD2 or CJ9-gD. The spinal column was cut into 4 pieces and eachpiece was kept separately in 0.5 ml of normal growth medium and storedat −80° C. for further processing. Total DNA was isolated from eachdorsal root ganglion using the DNeasy tissue kit (Qiagen, Santa Clarita,Calif.), and suspended in 400 μl AE buffer. The presence of HSV-2 DNAwas quantified by real-time PCR (Applied Biosystems 7300 Real-Time PCRSystem) with 100 ng of ganglia DNA and primers specific to the HSV DNApolymerase (Forward: 5′ GCT CGA GTG CGA AAA AAC GTT C (SEQ ID NO:4),Reverse: 5′ CGG GGC GCT CGG CTA AC (SEQ ID NO:5)) as previouslydescribed (8). The minimal copies of HSV-2 viral DNA that can bereliably detected were 1 copy per reaction.

Statistical Analysis

For statistical analysis un-paired Student's t-tests were performed.Results are considered to be statistically significant when the P valueis less than 0.05.

II. Results

Construction of CJ2-gD2

As the first step in generating a gD2- and UL9-C535C-expressingdominant-negative and replication-defective HSV-2 recombinant virus, weconstructed an HSV-2 ICPO deletion mutant, N2-lacZ, in which both copiesof ICP0 gene in HSV-2 strain 186 are replaced by the LacZ gene under thecontrol of the HSV-2 ICP0 promoter (FIG. 1B). We show that, similar tothe HSV-1 ICP0 null mutant 7314 (11), the plaque-forming efficiency ofN2-lacZ on human osteosarcoma line U2OS cells is 425-fold higher than inVero cells, indicating that the cellular activity in U2OS cells can alsofunctionally substitute for HSV-2 ICP0. Compared with wild-type HSV-2,replication efficiency of N2-lacZ in Vero cells is reduced over 600-foldat an MOI of 0.1 PFU/cell. Consistent with this finding, intravaginalinoculation of N2-lacZ at 1×10⁵ and 5×10⁵ PFU/mouse led to no local orsystemic illness, while mice infected with 1×10⁴ PFU/mouse of wild-typeHSV-2 developed severe genital herpes, and all died by day 11post-infection. Moreover, N2-lacZ fails to establish reactivatablelatent infection following intravaginal infection at a dose of 5×10⁵PFU/mouse. These results indicate that, similar to HSV-1 ICP0 (10, 11,37, 64), deletion of HSV-2 ICP0 significantly impairs the ability of thevirus to initiate acute and reactivatable latent infection in vivo.

Aiming to maximize levels of gD2 expression by a dominant-negative andreplication-defective HSV-2 viral recombinant, we constructed adominant-negative and replication-defective HSV-2 recombinant (CJ2-gD2)by replacing both copies of the Lac Z gene in N2-lacZ with DNA sequencesencoding the gD2 gene driven by the tetO-bearing HSV-1 majorimmediate-early ICP4 promoter and UL9-C535C under control of thetetO-containing hCMV major immediate-early promoter with a truncation atthe −236 bp of the full-length of hCMV immediate-early promoter (FIG.1B). Thus, unlike CJ9-gD, which encodes a single copy of the insertedHSV-1 gD gene driven by the tetO-containing hCMV promoter at the HSV-1UL9 locus (41), CJ2-gD2 contains 2 copies of gD2 gene controlled by thetetO-bearing HSV-1 immediate-early ICP4 promoter, which consists ofHSV-1 ICP4 promoter sequence from −377 bp to −19 bp relative to thetranscriptional start site of ICP4 gene. N2-C535C is an HSV-2recombinant in which both copies of the Lac Z gene in N2-lacZ arereplaced by UL9-C535C under the control of the full-length tetO-bearinghCMV immediate-early promoter.

CJ2-gD2 Expresses High Levels of gD2 and UL9-C535C in Infected VeroCells

To examine expression of gD2 and UL9-C535C from the tetO-bearing HSV-1immediate-early ICP4 promoter and hCMV immediate-early promoter,respectively, Vero cells were infected with wild-type HSV-2, N2-lacZ,N2-C535C, and CJ2-gD2 at a MOI of 10 PFU/cell and harvested at 9 hpost-infection. Infected cell proteins were analyzed by western blotassays with an HSV-1/2 ICP27 monoclonal antibody, a UL9 polyclonalantibody, and a gD1 polyclonal antibody (R45). Given that, like gD2, gB2is the major target for neutralizing antibody as well as T-cellresponses and is a γ1 product, infected cell proteins were also probedwith a gB-specific monoclonal antibody. FIG. 2A shows that CJ2-gD2 andN2-C535C express similar levels of HSV-2 immediate-early protein ICP27to those expressed by wild-type HSV-2 and N2-lacZ. While significantamounts of UL9-C535C were detected in CJ2-gD2- and N2-C535C-infectedcells, little gD2 or gB2 was detected in N2-C535C-infected cells. Incontrast to N2-C535C infection, however, infection of Vero cells withCJ2-gD2 leads to high-level expression of gD2 at levels similar to thosein cells infected by wild-type HSV-2, and gD2 expression has no effecton gB2 expression. The results also indicate that, like the HSV-1 ICP0null mutant 7134 (71), deletion of HSV-2 ICP0 in N2-lacZ greatly reducesgD2 expression. Due to the very low-level expression of UL9 from itsauthentic HSV early promoter (68), no wild-type UL9 was detected amongcells infected by these four different viruses. Additionally, we observethat levels of UL9-C535C expressed in CJ2-gD2-infected cells areconsistently higher than in cells infected by N2-C535C, suggesting thatthe HSV VP16 responsive elements, TAATGARAT, present in the HSV-1 ICP4promoter (71) can lead to enhanced expression of UL9-C535C from thehCMV-immediate-early promoter of the described hybrid ICP4/hCMV promotersystem.

Western blot analysis with the gD1 polyclonal antibody (R45) presentedin FIG. 2B shows that while much higher levels of gD were detected inwild-type HSV-1-infected cells than in cells infected with wild-typeHSV-2, levels of gD detected in CJ9-gD-infected cells were markedlylower than in cells infected by CJ2-gD2. This finding demonstrates thatCJ2-gD2 expresses gD2 more efficiently than gD1 expressed by CJ9-gD.

To demonstrate that the UL9-C535C and gD2 expressed in CJ2-gD2-infectedVero cells are indeed under the control of the tetO-bearing promoters,we next infected a stable tetR-expressing Vero cell line, VCEP4R-28cells, with wild-type HSV-2 and CJ2-gD2 at an MOI of 10 PFU/cell in theabsence or presence of tetracycline. Proteins from infected cells wereharvested at 9 h post-infection and analyzed by western blots. As can beseen (FIG. 3), although similar levels of ICP27 were detected inwild-type HSV-2- and CJ2-gD2-infected VCEP4R-28 cells in both theabsence and presence of tetracycline, UL9-C535C was detected inCJ2-gD2-infected VCEP4R-28 cells only when tetracycline was present andsignificantly higher levels of gD2 were detected in the presence oftetracycline than in its absence.

CJ2-gD2 Cannot Replicate in Vero Cells

Because of the lack of ICP0 and high-level expression of UL9-C535C fromthe tetO-bearing hCMV major immediate-early promoter, CJ2-gD2 had to beconstructed and propagated in the tetR-expressing ICP0 complementingU2OS cell line U2CEP4R11 (68). We plaque-assayed 6.65×10⁷ PFU of CJ2-gD2on Vero cell monolayers and detected no infectious virus, demonstratingthat the plaque-forming efficiency of CJ2-gD2 in Vero cells is reducedat least 6.65×10⁷-fold compared with its complementing U2CEP4R11 cells.

Inhibition of Wild-type HSV-2 Replication by CJ2-gD2

We next tested the dominant-negative effect of high-level UL9-C535Cexpression by CJ2-gD2 on the replication of wild-type HSV-2 viralreplication by the co-infection assay (FIG. 4). FIG. 4A shows thatco-infection of Vero cells with CJ2-gD2 at a MOI of 5 PFU/cell andwild-type HSV-2 at an MOI of 2 PFU/cells led to a nearly 500-folddecrease in wild-type HSV-2 production compared with cells singlyinfected by wild-type HSV-2 at the same MOI, regardless of whether thevirus titers were determined in Vero cells or in U2CEP4R11 cells. Littlereduction in wild-type virus yield was detected when a similarco-infection experiment was performed with N2-lacZ.

To further examine the potency of CJ2-gD2 in inhibiting the replicationof wild-type HSV-2, we carried out co-infection experiments withwild-type HSV-2 and CJ2-gD2 at MOI ratios of 1:1 and 3:1, respectively.The results in FIG. 4B show that CJ2-gD2 is effective in preventingwild-type HSV-2 infection under both conditions, leading to about 151-and 94-fold reduction in wild-type virus synthesis at the indicatedco-infection ratios compared with cells singly infected with thewild-type HSV-2 at MOIs of 5 PFU/cell and 15 PFU/cell, respectively.

CJ2-gD2 is Avirulent Following Intracerebral Injection in Mice

Neurovirulence is one of the hallmarks of HSV infection. To determinethe ability of CJ2-gD2 and N2-C535C to replicate in the CNS, femaleBALB/c mice 5 to 6-weeks-old were randomly assigned to five groups of 8mice each. CJ2-gD2 and N2-C535C were directly inoculated into the brainof each mouse at the left frontal lobe at 2.5×10⁶ PFU per mouse in a 20μl volume with a 28-gauge insulin needle at the depth of 4 mm (74).Morbidity and mortality were monitored for 35 days. Given that the LD₅₀of wild-type HSV-2 strain 186 is around 10 PFU in female BALB/c miceafter intravitreal injection (38), a group of mice were also inoculatedwith wild-type HSV-2 at 25 PFU/mouse. As an additional control, mice inthe fifth group were inoculated with N2-lacZ at 1×10⁶ PFU/mouse. FIG. 5shows that, like mice inoculated with DMEM, mice intracerebrallyinoculated with CJ2-gD2 and N2-C535C at a dose of 2.5×10⁶ PFU showed nosigns of neurovirulence during a 35-day follow-up, while all miceinoculated with wild-type HSV-2 at a dose of 25 PFU/mouse (a100,000-fold lower dose than that given to mice inoculated with CJ2-gD2)died by day 10 post-inoculation, and all inoculated mice exhibited signsof CNS illness commonly associated with HSV-2 infection, includingroughened fur, hunched posture, ataxia, and anorexia. Although 100% ofmice inoculated with N2-lacZ survived, all mice exhibited signs ofencephalitis.

Induction of HSV-2-Specific Neutralizing Antibodies and a gD2-Specific

Antibody Response in Mice Immunized with CJ2-gD2

The ability of CJ2-gD2 to elicit anti-HSV-2-specific neutralizingantibodies was determined in mice immunized with CJ2-gD2 at a dose of2×10⁶ PFU. As controls, groups of mice were also immunized with N2-C535Cor CJ9-gD at the same dose. As shown (FIG. 6A), the average of theHSV-2-specific neutralization antibody titer in mice immunized withCJ2-gD2 were on average 500, which is 3-fold higher than that of miceimmunized with N2-C535C (p=0.015), and is comparable to the neutralizingantibody titer induced in CJ9-gD immunized mice (p=0.28). No specificantibody titers against HSV-2 were detected in mock-vaccinated mice at a1:10 dilution.

FIG. 6B shows that while similar levels of gD-specific antibody responsewere detected between mice immunized with CJ2-gD2 and CJ9-gD when therespective immunoprecipitated gD2 complexes were probed with anti-gD1antibodies, R45, levels of anti-gD-specific antibodies in mice immunizedwith CJ2-gD2 were significantly higher than in mice immunized withN2-C535C and mock-immunized control. Taken together, the resultspresented in FIG. 6 indicate that high-level expression of gD2 bpCJ2-gD2 leads to increased efficacy in eliciting anti-gD2 antibody aswell as anti-HSV-2-specific neutralizing antibody responses comparedwith N2-C535C.

Induction of HSV-2-Specific T-Cell Response in Mice Immunized withCJ2-gD2

To evaluate the effectiveness of CJ2-gD2 immunization in elicitingHSV-2-specific T-cell response, we carried out the recall experiment toexamine the memory T-cell responses in immunized mice followingchallenge with wild-type HSV-2. First, sham-vaccinated andCJ2-gD2-vaccinated mice were either mock-challenged or challenged withwild-type HSV-2 at 9-10 weeks post-boost immunization followed by IFN-γELISPOT assays with CD4⁺ and CD8⁺ T cells isolated from spleens ofindividual groups of mice (n=3) on day 5 post-challenge (FIG. 7A).CJ2-gD2-vaccinated mice challenged with wild-type HSV-2 had a 4.8-foldincrease of IFN-γ-positive CD4⁺ T cells compared with the mock-infectedCJ2-gD2-immune mice (p<0.0001). More significantly, the number ofIFN-γ-secreting CD4⁺ T cells detected in HSV-2-infected mice previouslyvaccinated with CJ2-gD2 was 18-fold more than HSV-2-infectedsham-vaccinated mice (p<0.0001). No IFN-γ-positive CD4 ⁺ T cells weredetected in sham-vaccinated mock-infected control mice under identicalconditions. These findings show that immunization with CJ2-gD2 elicitsstrong memory CD4⁺ T cell response.

While there was a greater than 2-fold increase in IFN-γ-secreting CD8⁺ Tcells in CJ2-gD2-vaccinated mice compared with the sham-vaccinatedcontrols, similar numbers of IFN-γ-secreting CD8⁺ T cells were detectedin the spleens of HSV-2-infected sham-vaccinated mice and HSV-2-infectedCJ2-gD2-vaccinated mice (FIG. 7B). We thus carried out the second set ofrecall experiments, in which sham-vaccinated and CJ2-gD2-vaccinated micewere either mock-challenged or challenged with wild-type HSV-2 (n=3) 5-6weeks post-second vaccination. CD4⁺ and CD8⁺ ELISPOT assays wereperformed on day 4 post-infection (FIGS. 7C and 7D). An 8.6- and5.7-fold increase in IFN-γ-secreting CD4⁺ and CD8⁺ T cells,respectively, was detected in CJ2-gD2 immune mice following HSV-2infection compared with mock-infected CJ2-gD2 immune mice (CD4 T cells:p=0.035; CD8⁺ T cells: p=0.01). Moreover, following challenge withHSV-2, IFN-γ-secreting CD4⁺ and CD8⁺ T cells were 8- and 9.5-foldhigher, respectively, in CJ2-gD2 vaccinated compared withsham-vaccinated mice (CD4⁺ T cells: p=0.036; CD8⁺ T cells: p=0.01).Collectively, these studies demonstrate that immunization with CJ2-gD2can elicit robust HSV-2-specific memory CD4⁺ and CD8⁺ T-cell responses,which can be efficiently recalled during HSV-2 infection.

Protection Against HSV-2 Genital Infection and Disease in Immunized Mice

Five to six weeks after the initial immunization, mice were challengedintravaginally with HSV-2 strain G at 50 LD₅₀ (5×10⁵ PFU/mouse). Vaginalswabs were taken on days 1, 2, 3, 5, and 7 after challenge. Mice wereobserved during a 21-day follow-up period for the incidence of genitaland disseminated HSV-2 disease. As shown in FIG. 8A, yields of challengevirus were reduced more than 200-fold on day 1 (p<0.001) and 130-fold onday 2 (p<0.0001) in mice immunized with CJ2-gD2 (n=9) compared withthose of mock-immunized control (n=10). Although there was nosignificant difference in reduction of challenge virus shedding on days1, 2, and 3 post-challenge between groups of mice immunized with CJ2-gD2and N2-C535C (n=10), immunization with CJ2-gD2 was more effective thanCJ9-gD in reducing challenge virus shedding on days 1 (p=0.03), 2(p=0.025), and 3 (p<0.007). Little or no challenge virus was detected inmice immunized with CJ2-gD2, N2-C535C, or CJ9-gD on day 5post-challenge, whereas all mock-vaccinated mice continued to shed virusat an average yield of more than 5×10³ PFU/ml. No challenge virus waspresent in the vaginal swab materials collected on day 7 post-challengein three immunized groups of mice. In a separate experiment, we observedthat while there was no virus shedding in CJ2-gD2-immunized mice on day5 post-challenge, presence of wild-type HSV-2 was detected in 5 out of 7N2-C535C-immunized mice and 4 out of 7 CJ9-gD-immunized mice.

The results in FIG. 9 show that mice immunized with CJ2-gD2 werecompletely protected from development of local genital lesions andexhibited no signs of systemic disease after challenge with wild-typeHSV-2 (FIG. 9A). All mock-immunized mice developed severe genitallesions and succumbed to the wild-type HSV-2 infection by day 11post-challenge (FIG. 9B). Although immunization with N2-C535C and CJ9-gDprotected mice against lethal challenge with wild-type HSV-2, 20% and30% of mice experienced a transient low degree of local genital disease(score 1) in N2-C535C- and CJ9-gD-immunized mice, respectively (Table1). In a similar experiment (Table 1), it was observed that among miceimmunized with CJ9-gD (n=7), 2 mice experienced low degrees of localgenital disease, and 1 mouse showed sign of systemic illness and died onday 14 post-challenge, and 3 out of 7 N2-C535C-immunized mice (43%)showed a low degree of local genital disease (score=1). Again, no signsof local and systemic herpetic disease were seen in CJ2-gD2-immunizedmice (n=7). Collectively, these studies demonstrate that CJ2-gD2 is amore effective vaccine than N2-C535C and CJ9-gD in protection miceagainst genital disease following intravaginal challenge with wild-typeHSV-2.

TABLE 1 Percentage of protection against herpetic disease inmock-immunized and immunized mice following intravaginal challenge withwild-type HSV-2 Mock CJ2-gD2 N2-C535C CJ9-gD Exp 1 (n = 9-10) 0 100% 80%70% Exp 2 (n = 7-8) 0 100% 57% 57%

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All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by those ofskill in the art that the invention may be practiced within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

1. A replication-defective, dominant-negative Herpes simplex virus 2(HSV-2) recombinant virus, comprising within its genome: a) a firstsequence encoding a first HSV-2 glycoprotein D (gD2) wherein saidsequence is operably linked to a first promoter and said first promoteris operably linked to a first tetracycline operator (Tet-O) sequence; b)optionally, a second sequence encoding a second HSV-2 gD2 wherein saidsecond sequence is operably linked to a second promoter and said secondpromoter is operably linked to a second tet-O sequence; c) a thirdsequence encoding a first dominant negative mutant form of HSV-1 orHSV-2 UL9 protein, wherein said third sequence is operably linked to athird promoter and said third promoter is operably linked to a thirdtetracycline operator (Tet-O) sequence; d) optionally, a fourth sequenceencoding a second dominant negative mutant form of HSV-1 or HSV-2 UL9protein, wherein said fourth sequence is operably linked to a fourthpromoter and said fourth promoter is operably linked to a fourthtetracycline operator (Tet-O) sequence; and wherein said genome does notcomprise a sequence encoding a functional ICP0 protein.
 2. Therecombinant virus of claim 1, wherein said recombinant virus compriseswithin its genome said second sequence encoding a second HSV-2 gD2wherein said second sequence is operably linked to a second promoter andsaid second promoter is operably linked to a second tet-O sequence. 3.The recombinant virus of claim 2, wherein said recombinant viruscomprises within its genome said fourth sequence encoding a seconddominant negative mutant form of HSV-1 or HSV-2 UL9 protein, whereinsaid fourth sequence is operably linked to a fourth promoter and saidfourth promoter is operably linked to a fourth tetracycline operator(Tet-O) sequence.
 4. The recombinant virus of claim 1, wherein one ormore of said first, second, third and fourth promoters is an hCMVimmediate early promoter.
 5. The recombinant virus of claim 1, whereinone or more of said first, second, third and fourth promoters is anHSV-1 or HSV-2 immediate early promoter.
 6. The recombinant virus ofclaim 5, wherein said HSV-1 or HSV-2 immediate early promoter is ICP4.7. The recombinant virus of claim 5, wherein said first, second, thirdand fourth promoters are all HSV-1 or HSV-2 immediate early promoters.8. The recombinant virus of claim 5, wherein said first, second, thirdand fourth promoters are ICP4 promoters.
 9. The recombinant virus ofclaim 1, wherein: a) said first, second, third and fourth promoters eachhave a TATA element; b) each of said first, second, third and fourthTet-O sequences comprise two op2 repressor binding sites joined by 2-20linking nucleotides, wherein the first nucleotide in said tet operatoris between 6 and 24 nucleotides 3′ to the last nucleotide in said TATAelement; c) said sequence encoding an HSV-2 gD2 and said second sequenceencoding an HSV-2 gD2 lie 3′ to said first and second Tet-O sequencesand are operably linked to said first and second promoters; d) saidsequence encoding said first dominant negative mutant form of HSV-1 orHSV-2 UL9 protein lies 3′ to said third Tet-O sequence and is operablylinked to said third promoter; e) said sequence encoding said seconddominant negative mutant form of HSV-1 or HSV-2 UL9 protein lies 3′ tosaid fourth Tet-O sequence and is operably linked to said fourthpromoter.
 10. The recombinant virus of claim 1, wherein said mutant formof UL9 protein is UL9-C535C.
 11. The recombinant virus of claim 1wherein said recombinant virus also expresses one or more recombinantimmunomodulating genes.
 12. The recombinant virus of claim 11, whereinsaid recombinant virus expresses IL12.
 13. The recombinant virus ofclaim 11, wherein said recombinant expresses IL15.
 14. The recombinantvirus of claim 1, wherein said recombinant virus also expresses HSV-2 gBunder the control of the tetO-bearing HSV or hCMV immediate-earlypromoter.
 15. The recombinant virus of claim 1, wherein said recombinantvirus also expresses HSV-2 gC under the control of the tetO-bearing HSVor hCMV immediate-early promoter.
 16. A vaccine comprising therecombinant virus of claim 1 in unit dose form.
 17. The vaccine of claim16, wherein said recombinant virus is present at a minimum of 1×10⁷ pfuper unit dose.
 18. The vaccine of claim 16, wherein said recombinantvirus is present at 1×10⁷-1×10⁹ pfu per unit dose.
 19. A method ofimmunizing a patient against HSV-1 or HSV-2 infection, comprisingadministering to said patient the vaccine of claim
 16. 20. The method ofclaim 19, wherein said patient is seropositive for either HSV-1 orHSV-2. 21-23. (canceled)